Alzheimer's Disease (AD) is a degenerative brain disorder associated with extensive loss of specific neuronal cellular subpopulations, and characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death. The disease currently affects as many as four million individuals in the United States alone. To date, the disease has proven to be incurable, and presently causes up to 100,000 deaths annually.
The brains of individuals with AD exhibit neuronal degeneration and characteristic lesions variously referred to as amyloidogenic plaques, vascular amyloid angiopathy, and neurofibrillary tangles. It is presently believed that progressive cerebral deposition of particular amyloidogenic proteins, beta-amyloid proteins, play a seminal role in the pathogenesis of AD and can precede cognitive symptoms and onset of dementia by years or possibly even decades.
Alzheimer's disease is associated with aberrant processing of the amyloid precursor protein (APP), leading to increased production and aggregation of amyloid-β (Aβ) peptides. Amyloid plaques are composed primarily of 40 and 42 amino acid peptides (Aβ40 and Aβ42, respectively) (Selkoe, Proc. Nat'l. Acad. Sci. USA 98:11039-11041 (2001)) derived from APP by sequential proteolysis catalyzed by the aspartyl protease, BACE (Vassar et al., Science 286:735-741 (1999)), followed by presenilin-dependent γ-secretase cleavage (De Strooper et al., Nature 391:387-390 (1998)). Aβ42 is less soluble than Aβ40 and is the predominant Aβ species in amyloid plaques (Iwatsubo et al., Neuron 13:45-53 (1994)).
Presenilins 1 and 2 (PS1 and PS2) are integral membrane proteins proposed to have inherent γ-secretase activity (Wolfe et al., Nature 398:513-517 (1999)) and interact in a functional complex with nicastrin (Esler et al., Proc. Nat'l. Acad. Sci. USA 99:2720-2725 (2002); Edbauer et al., Proc. Nat'l. Acad. Sci. USA 99:8666-8671 (2002)), aph-1, and pen-2 (Francis et al., Dev. Cell 3:85-97 (2002)). Presenilins also interact with a number of other proteins, including α-catenin and β-catenin (Soriano et al., J. Cell Biol. 152:785-794 (2001); Yu et al., Nature 407:48-54 (2000)). Presenilin 1, which is required for γ-secretase mediated processing of APP (De Strooper et al., 1998), interacts with glycogen synthase kinase-3 (GSK-3)(Takashima et al., Proc. Nat'l. Acad. Sci. USA 95:9637-9641 (1998); Kang et al., J. Neurosci. 19:4229-4237 (1999); Kang et al., Cell 110:751-762 (2002)), although a functional role for this proteins in γ-secretase function has not been previously established.
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase having a monomeric structure and a size of approximately 47 kilodaltons. It is one of several protein kinases which phosphorylate glycogen synthase (Embi et al., Eur. J. Biochem. 107:519-527 (1980); Hemmings et al., Eur. J. Biochem. 119:443-451 (1982)). GSK-3 is also referred to in the literature as factor A (FA) in the context of its ability to regulate FC, a protein phosphatase (Vandenheede et al., J. Biol. Chem. 255:11768-11774 (1980)). Other names for GSK-3 and homologs thereof include: zeste-white3/shaggy (zw3/sgg; the Drosophila melanogaster homolog), ATP-citrate lyase kinase (ACLK or MFPK; Ramakrishna et al., Biochem. 28:856-860 (1989); Ramakrishna et al., J. Biol. Chem. 260:12280-12286 (1985), GSKA (the Dictyostelum homolog; Harwood et al., Cell 80:139-48 (1995), and MDSI, MCK1, and others (yeast homologs; Hunter et al., TIBS 22:18-22 (1997)), tau protein kinase (mammalian) and GSKA (Dictyostelium).
The gene encoding GSK-3 is highly conserved across diverse phyla. In vertebrates, GSK-3 exists in two isoforms, designated GSK-3α (51 kDa) and GSK-3β (47 kDa). These two isoforms are the products of distinct genes. The amino acid identity among vertebrate homologs of GSK-3 is in excess of 98% within the catalytic domain (Plyte et al., Biochim. Biophys. Acta 1114:147-162 (1992)), although GSK-3α is known to be slightly larger than GSK-3β.
Sun et al., J. Biol. Chem. 277(14):11933-11940 (April 2002) have reported that in brain extracts and in MAP fractions, the amounts of GSK-3α and GSK-3β are almost equal, but that there are profound differences between the amounts of each kinase complexed with tau, further distinguishing the functions of the two. The authors determined that 6-fold more tau is complexed with GSK-3β than with GSK-3α in the brain, and that GSK-3β is bound to tau within an approximately 400-kDa micro-tubule-associated complex. Thus, GSK-3β associates with the microtubules via tau.
GSK-3 phosphorylates numerous proteins in vitro, including beta-catenin, glycogen synthase, phosphatase inhibitor I-2, the type-II subunit of cAMP-dependent protein kinase, the G-subunit of phosphatase-1, ATP-citrate lyase, acetyl coenzyme A carboxylase, myelin basic protein, a microtubule-associated protein, a neurofilament protein, an N-CAM cell adhesion molecule, nerve growth factor receptor, c-Jun transcription factor, JunD transcription factor, c-Myb transcription factor, c-Myc transcription factor, L-myc transcription factor, adenomatous polyposis coli tumor suppressor protein, and tau protein (Plyte et al., 1992; Korinek et al., Science 275:1784-1787 (1997); Miller et al., Genes & Dev. 10:2527-2539 (1996)). The phosphorylation site recognized by GSK-3 has been determined in several of these proteins (Plyte et al., 1992). The diversity of these proteins suggests a wide role for GSK-3 in the control of cellular metabolism, regulation, growth, and development. GSK-3 tends to phosphorylate serine and threonine residues in a proline-rich environment, but does not display the absolute dependence upon these amino acids which is displayed by protein kinases which are members of the mitogen-activated protein (MAP) kinase or cdc2 families of kinases.
U.S. Pat. No. 6,441,053 (Klein et al.) teaches a method of identifying inhibitors of GSK-3 and for treating a GSK-3-related disorders—other than Alzheimer's disease in an animal. The method comprises providing a mixture comprising GSK-3, a phosphate source, and a GSK-3 substrate, incubating the mixture in the presence or absence of a test compound, and assessing the activity of GSK-3 in the mixture. A reduction of GSK-3 activity following incubation of the mixture in the presence of the test compound is an indication that the test compound is an inhibitor of GSK-3. In the '053 patent, however, the GSK-3 inhibitor is expressly not lithium.
U.S. Pat. No. 6,057,117 (Harrison et al.) teaches a pharmaceutical composition comprising a selective GSK-3 inhibitor identified by: (a) contacting a first radiolabeled peptide substrate comprising an isolated nucleotide sequence, in which the N-terminal serine is the target of phosphorylation by GSK-3 and the C-terminal serine is prephosphorylated, coupled to an anchor ligand with GSK-3 in the presence of radiolabeled phosphate-γATP, a substrate anchor, and a candidate inhibitor, then (b) contacting a second radiolabeled peptide substrate coupled to an anchor ligand with GSK-3 in the presence of radiolabeled phosphate-γATP, and a substrate anchor, and (c) identifying an inhibitor of GSK-3 kinase activity by a reduction of radiolabel incorporation in step (a) compared to step (b). The identified composition is also used to treat a subject having a condition mediated by GSK-3 activity or susceptible to such a condition. In an alternative embodiment of the '117 patent a second therapeutic compound may be added, wherein the compound may be lithium. However, no lithium therapy is suggested with regard to blocking or inhibiting the activity of the GSK-3α isoform. See also Stambolic et al., Current Biology 6(12): 1664-1668 (1996).
The activity of both GSK-3α and -3β has been reported to be inhibited by lithium (e.g., Klein et al., Proc. Natl. Acad. Sci. USA 93:8455-8459 (1996); Hedgepeth et al., Dev. Biol. 185:82-91(1997); Phiel et al., Annu. Rev. Pharmacol. Toxicol. 41:789-813 (2001); U.S. Publ. Patent Appl. 20010052137 (Klein et al.)), yet specific inhibitors of the activity of the GSK-3α isoform alone (without affecting GSK-3β) remain unknown. Inhibition of GSK-3β is a physiological mechanism by which lithium exerts its therapeutic effects in animals (e.g., humans) afflicted with a variety of disorders. For example, lithium is an effective drug for treatment of bipolar (manic-depressive) disorder (Price et al., New Eng. J. Med. 331:591-598 (1994); Goodwin et al., (1990) In: Manic-Depressive Illness, New York: Oxford University Press), and can be used to treat profound depression in some cases, although it is not known whether lithium works through GSK-3 in the treatment of bipolar disorder. Despite the remarkable efficacy of lithium observed during decades of its use, the molecular mechanism(s) underlying its therapeutic actions have not been fully elucidated (Bunney et al., (1987) In: Psychopharmacology: The Third Generation of Progress, (Hy, ed.) New York, Raven Press, 553-565; Jope et al., Biochem. Pharmacol. 47:429-441 (1994); Risby et al., Arch. Gen. Psychiatry 48:513-524 (1991); Wood et al., Psychol. Med. 17:570-600 (1987)).
Lithium is a fixed monovalent cation and the lightest of the alkali metals (group 1a of the Periodic Table of the elements). Li+ has the highest energy of hydration of the alkali metals and, as such, can substitute for Na+ (and to a lesser extent K+) for ion transport by biological systems. Lithium is both electroactive and hydrophilic, and trace amounts of Li+ are found in human tissues; typical human blood plasma concentrations of Li+ are about 17 μg/L.
Unlike other psychotropic drugs, Li+ has no discernible psychotropic effects in normal man, although the therapeutic efficacy of lithium in the treatment of acute mania and the prophylactic management of bipolar (manic/depressive) disorder has been consistently demonstrated. The oral and parenteral administration of lithium salts, such as lithium carbonate and lithium citrate, has also found widespread use in the current treatment of, for example, alcoholism, aggression, schizophrenia, unipolar depression, skin disorders, immunological disorders, asthma, multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, and irritable bowel syndrome, as well as for use in many other diseases and conditions.
Unfortunately, no drug treatments for Alzheimer's disease have, to date, proven to be very satisfactory, and demonstrating the effectiveness of such drugs in the treatment of dementias can be quite difficult, see, e.g., Handbook of Dementing Illnesses, (John Morris, Ed.), Marcel Dekker 1994, p. 591. Part of this difficulty arises from the fact that it can often be difficult to clearly diagnose the type of dementia with which the patient is afflicted. Thus, there exists a pressing need to identify compositions that have a blocking or inhibiting effect in humans on the control of GSK-3α specifically required for APP processing to (Aβ) peptides, and/or to reduce formation of both amyloid plaques and neurofibrillary tangles, recognized as two pathological hallmarks of Alzheimer's disease. By finding a GSK-3α-specific inhibitor, preferably that does not also affect GSK-3β, it will be possible to treat Alzheimer's disease in a patient without inhibiting GSK-3β, which serves many critical functions in cells.